Cryptococcus neoformans is a fungal pathogen that infects the human brain. The organism has a defined sexual cycle involving haploid MATalpha and MATa strains, and the MATalpha locus has been linked to virulence and differentiation. Most environmental and clinical isolates are MATalpha mating type, and MATalpha strains are more virulent than congenic MATa strains. MATalpha strains also haploid fruit and produce filaments and infectious basidiospores when nitrogen limited. Thus, the structure and function of the mating type loci and their links to virulence and ecology are of considerable interest. Moore and Edman used difference cloning to identify part of the MATalpha locus encoding a mating pheromone. Later, Wickes and colleagues identified a Ste12 homolog that is MATalpha-specific but not encoded by the known MATalpha region. STE12alpha is required for virulence of a congenic lab adapted serotype D strain but is dispensable for virulence of a pathogenic serotype A clinical isolate. To establish functions of the mating type loci, we propose to clone, sequence, and mutate the MAT loci from three divergent C. neoformans varieties. MATalpha and MATa strains of each variety were known or identified in our lab and MATalpha and MATa specific genes were cloned. Six BAC libraries were constructed to isolate clones spanning each MAT locus. Sequencing of the serotype A and D MATalpha and MATa loci is in progress. These loci are unusually large (greater than 100 kb) with more than a dozen genes encoding three pheromones and a pheromone receptor, homologs of Ste20, Stel1, Ste12, and a Zn2+ finger protein. Other genes encode a myosin, a favoprotein, and a translation factor. Our studies reveal the MATalpha and MATa loci encode diverged alleles of the same genes and have extensively rearranged during evolution. Mutation of individual MATalpha locus genes confers defects in mating, fruiting, and virulence. A haploid strain lacking a 50 kb region of the MATalpha locus was inviable, indicating one or more genes in the region is essential. A diploid a/delta strain deleted for this part of the MATalpha locus was still self-filamentous like an a/alpha diploid, and a/a and alpha/alpha diploids were not self- filamentous, excluding a ploidy-dependent model. Our preliminary results support a locus-dependent model. We have discovered a novel, linked region of the MATalpha locus encoding a homeodomain homolog (Hdp1alpha) and a candidate transcriptional regulator (Rum1alpha), and we will establish the functions of these genes. We also propose to develop DNA based methods to test if MAT locus alterations occur naturally and affect fertility or virulence. These studies will provide insights into the role of MAT loci and sexual cycles in fungal differentiation and virulence.